Combining Asymmetric PCR with PAM-Independent Cas12a Analysis of Single-Stranded Amplicons for On-Site Nucleic Acid...

A biosensing system relying on asymmetric PCR (aPCR) and PAM-independent recognition of single-stranded DNA amplicons by a Cas12a/gRNA complex is adapted for on-site nucleic acid testing (NAT). aPCR was performed on a low-cost, portable, smartphone-controlled thermocycler operating in endpoint mode. Subsequently, a selective visual readout of the aPCR outcome was achieved using a Cas12a-based assay. A mechanism to prevent false positives was incorporated into the aPCR/Cas12a biosensing system by introducing uracil into the amplicons produced during amplification. Pectobacterium species-harmful bacterial plant pathogens-and genus-specific primers Y1 and Y2 against Pectobacterium spp. were utilized as a convenient model. The absence of a PAM requirement allowed for fine-tuning the selectivity of the Cas12a assay against a strain of a particular Pectobacterium species, P. polaris, by properly positioning the gRNA spacer on the ssDNA. P. polaris was detected down to 10 copies of the bacterial genome per reaction, with an overall testing time of about 2 h and either instrumental or visual readouts. Similar selectivity and sensitivity toward a P. polaris strain were observed with the aPCR/Cas12a biosensing system for potato samples artificially contaminated with Pectobacterium species. The overall findings indicate aPCR/Cas12a-based biosensing as a technique suitable for on-site NAT.